BET Protein Function Is Required for Inflammation: Brd2 Genetic Disruption and BET Inhibitor JQ1 Impair Mouse Macrophage Inflammatory Responses

Abstract

Histone acetylation regulates activation and repression of multiple inflammatory genes known to play critical roles in chronic inflammatory diseases. However, proteins responsible for translating the histone acetylation code into an orchestrated proinflammatory cytokine response remain poorly characterized. Bromodomain and extraterminal (BET) proteins are "readers" of histone acetylation marks, with demonstrated roles in gene transcription, but the ability of BET proteins to coordinate the response of inflammatory cytokine genes through translation of histone marks is unknown. We hypothesize that members of the BET family of dual bromodomain-containing transcriptional regulators directly control inflammatory genes. We examined the genetic model of brd2 lo mice, a BET protein hypomorph, to show that Brd2 is essential for proinflammatory cytokine production in macrophages. Studies that use small interfering RNA knockdown and a small-molecule inhibitor of BET protein binding, JQ1, independently demonstrate BET proteins are critical for macrophage inflammatory responses. Furthermore, we show that Brd2 and Brd4 physically associate with the promoters of inflammatory cytokine genes in macrophages. This association is absent in the presence of BET inhibition by JQ1. Finally, we demonstrate that JQ1 ablates cytokine production in vitro and blunts the "cytokine storm" in endotoxemic mice by reducing levels of IL-6 and TNF-a while rescuing mice from LPS-induced death. We propose that targeting BET proteins with small-molecule inhibitors will benefit hyperinflammatory conditions associated with high levels of cytokine production. The Journal of Immunology, 2013, 190: 000-000. R egulation of inflammatory gene expression is tightly controlled through chromatin "readers" that specifically bind histone posttranslational modifications and provide a scaffold, which, in addition to sequence-specific transcription factors, is an integral component of the transcriptional activation complex (1). The biological effects of chromatin-dependent, multiprotein complexes include both transcriptional coactivation and corepression of inflammatory genes in differentiated adult cells (2); thus chromatin readers play critical roles in exquisitely tuned inflammatory responses to a variety of immune system stimuli. Proinflammatory stimuli such as bacterial endotoxin (LPS) arouse extensive transcriptional reprogramming through their ability to activate acetylation of ε-amino groups of nucleosomal histone lysines, a general mark of gene activation (3-5). The acetylated lysine residues are recognized by chromatin readers, many of which contain a conserved structure designated the bromodomain. Bromodomains are highly conserved, left-twisted bundles of four a-helices, with a hydrophobic cleft between two conserved loops that connect the helices (6). The motif uses hydrogen bonding, often at asparagine residues, to bind to acetylated histones (7). In humans, there are at least 40 bromodomain proteins (8, 9), which include histone acetyltransferases, helicases, scaffolding proteins, and other cofactors that control gene transcription. These findings raise the possibility that bromodomain proteins regulate acetylated, histone-packaged inflammatory genes through multiple downstream mechanisms to significantly contribute to outcomes from proinflammatory stimuli. The bromodomain and extraterminal domain (BET) family is a distinct group of bromodomain proteins that in mammals includes Brd2, Brd3, and Brd4, all of which are ubiquitously expressed in mammalian tissues (10-13). Brd2 and Brd4 have been extensively studied in the context of cell-cycle control (14-18) and transcription elongation (19-21), but potential roles in inflammatory responses have not been explored well. Establishing links between BET proteins and inflammation has become clinically critical owing in part to recent drug development efforts, which have shown that drugs able to interrupt interactions between Brd4 and thienodiazepines (22) have efficacy in BET protein-related cancers (23, 24). JQ1 was the first drug developed that specifically interacts with the hydrophobic pocket of the BET bromodomain to block interaction between multiple BET proteins (Brd2/3/4) and acetylated histones (9). JQ1 effectively prompted squamous differentiation and reduced tumor volume of Brd4-dependent, human NUT midline carcinoma xenografts in mice (9), and was proved efficient to block growth of various leukemic cell